The two pantothenate-contained proteins previously found by us in Neurospora mitochondria have now been identified by us as the acylated and unacylated forms of an ACP (acyl carrier protein). Since this is the first report of an ACP in the mitochondria of any organism and the first report of ACP in any non-photosynthetic eucaryotic cell, it is proposed to explore its function in the mitochondria as well as its coordination with the cytoplasmic multifuntional fatty acid synthetase. Initial studies on the nature of the acyl groups esterified to 6000 fold purified ACP indicate that the major one is 3-OH 14:0, a known intermediate in acyl chain elongation. Studies are proposed on: 1) the identification of any additional acyl moieties on the ACP; 2) in vitro labelling of the acyl groups of ACP in order to determine precursor-product relationships; 3) the primary structure of this ACP vs. the plant and bacterial ACP(s); 4) the possible presence of ACP in the mitochondria of other organisms; and 5) the mode and site of biosynthesis of ACP as well as its post-translational modifications. These experiments will also utilize two mutant strains: one known to be deficient in the cytoplasmic fatty acid synthetase (the cell strain) and a newly-isolated Ts saturated fatty acid-requirer. The selection of new mutants in the ACP pathway will also be attempted. These studies are viewed as basic studies of eucaryotic microbial physiology directed towards understanding membrane biosynthesis, the role of this particular pathway, and its reason(s) for being localized to the mitochondria. In addition, this system might be able to serve as a model system for studying the biosynthesis of the very long chain acyl groups found exclusively in sphingolipids, a class of compounds critical to nerve cell function, and whose biosynthesis in brain tissue occurs via some unknown activated form of fatty acids, CoA- independent, and with some involvement of the mitochondria.